Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by self-limited attacks of fever with serosal, synovial, or cutaneous inflammation, sometimes complicated by systemic amyloidosis. In 1992 our laboratory mapped the FMF locus to chromosome 16p13.3, and in 1997 we isolated the underlying gene, MEFV, and demonstrated that it is highly expressed in granulocytes. During the 7 years leading up to the present reporting period, we have focused on several areas, including FMF population genetics, the regulation of FMF gene expression in leukocyte subpopulations, the biochemistry and cell biology of pyrin (the FMF protein), and the development of animal models of FMF. In studies of a mouse strain expressing a truncated form of pyrin, we found that pyrin plays a major role in caspase-1 activation and IL-1beta processing, and that pyrin regulates macrophage apoptosis through a caspase-8-dependent, IL-1beta-independent pathway. By yeast two-hybrid studies, we demonstrated that pyrin interacts with the cytoskeletal protein PSTPIP1, and that PSTPIP1 mutations associated with the syndrome of pyogenic arthritis with pyoderma gangrenosum and acne (PAPA) lead to markedly increased pyrin-binding and IL-1beta activation. Finally, during the year directly antecedent to the present reporting period, we found that pyrin can be cleaved by caspase-1, and that the N-terminal cleavage fragment of pyrin activates NF-kappaB. Results of the Last Year Effects of pyrin on NFkappa B activation: A major focus of the last year has been to follow up on the observation that an N-terminal cleavage fragment of pyrin (aa 1 ? 330) activates NF-kappaB. We had previously also shown that FMF-associated pyrin mutants undergo increased caspase-1-mediated cleavage, relative to wild type (WT), and that absolute and relative amounts of cleaved pyrin are substantially increased in leukocytes from FMF patients compared with healthy controls. Biochemical analysis has now shown that the N-terminal cleavage fragment of pyrin interacts with IkappaBalpha and NF-kappaB p65, but that full length pyrin does not interact. Through a series of deletion mutants, we have found that pyrin interacts with these proteins through its bZIP basic domain (aa 266 ? 280). Moreover, we have found that the N-terminal fragment of pyrin both enhances both the degradation of IkappaBalpha and the entrance of NF-kappaB into the nucleus, perhaps explaining its NF-kappaB stimulatory activity. Gene expression profiling in pyrin-null mice: Prior to the present reporting period, we had generated pyrin-null mice by targeted disruption of exons 1 and 2 of murine Mefv, and backcrossed onto a C57BL/6 background. Similar to pyrin-truncation mice (see above), pyrin -/- animals were produced at the expected Mendelian frequency, developed normally, and reproduced as well as WT littermates. We studied the gene expression profile of resident peritoneal macrophages of male C57BL/6 pyrin -/- and WT mice. Labeled cRNA prepared from total RNA of peritoneal macrophages was hybridized to Affymetrix GeneChips interrogating 22690 transcripts. For statistical analysis we generated four-step filtering criteria. In both age groups of the pyrin -/- mice, 85 genes were found to be differentially expressed. The largest functional group of mRNAs that showed altered expression comprised genes involved in inflammation and the immune response. Consistent with our previous results for pyrin truncation mice, pyrin -/- mice showed an upregulation of several members of the IL-1 family, which was confirmed by Western blot for IL-1 beta. In addition, a number of other genes were upregulated, including cryopyrin, chemokines, Fc receptors, and genes involved in the stress response, further suggesting that reduction in pyrin activity leads to autoinflammation. Nevertheless, underlining the complexity of the regulatory network, genes involved in host defense to pathogens were downregulated relative to WT mice. Gene expression studies in FMF patients: In order to compare global gene expression patterns between FMF patients and healthy controls, we extracted mRNA from peripheral blood mononuclear cells from 26 mutation-positive FMF patients on colchicine therapy and 37 healthy controls. cRNA was subsequently hybridized the Affymetrix U133A DNA microarrays, and data were processed by two independent signal quantitation algorithms, RMA and MAS5.0. Preliminary analysis has identified several classes of genes that show decreased levels of expression in FMF patients relative to controls. These include genes involved in the regulation of the inflammatory response (CXCL5, CD69, and CD160), apoptosis (BCL2), and gene transcription (NR4A2, EGR1, and EGR3). Although not yet proven, it is possible that at least some of these genes are downregulated in patients due to colchicine therapy. There were also a small number of genes that were upregulated in FMF patients relative to controls, including those involved in host defense (MPO, DEFA4, DEFA1) and the inflammatory response (TNFA1P6). RNA interference in the study of pyrin function in man: In order to study the function of pyrin in human myeloid cells, we developed a model system involving the transfection of small interfering RNA (RNAi) into the THP.1 myeloid leukemia cell line. We found that MEFV siRNA can be transfected into THP.1 cells with an efficiency of over 75 percent. The high percentage of transfection led to a significant decrease in the endogenous levels of pyrin protein as compared with a scramble control siRNA (SC). To provide insight into pyrin function, we compared expression profiles between SC and siMEFV using microarray. We identified a total of 344 genes with a 1.5 or more fold difference and a p of less than 0.05, 252 of which were upregulated in the pyrin knockdown relative to SC, and 92 of which were upregulated relative to SC. To date two of the genes downregulated by MEFV siRNA have been validated by quantitative RT-PCR, S100A8 and CD36. Studies of FMF patients undergoing a structured treatment interruption of colchicine: Seven patients with mutation-positive FMF underwent a closely-monitored 3 ? 4 day interruption of colchicine therapy. Five of the seven patients had no clinical symptoms or change in the acute-phase reactants, while two reported an attack at 48 hours, with a significant elevation in at least one acute-phase reactant. Patients typically demonstrated a higher ratio of cleaved pyrin to full-length pyrin as compared with controls, and the proportion of cleaved pyrin was reciprocally related to levels of IkappaBalpha. A stepwise increase in neutrophil activation was seen during the first 48 hours, with no change in lymphocyte subpopulations. Conclusions and Significance Data obtained during the current reporting period corroborate earlier findings implicating pyrin as an important regulator of cytokine production, NF-kappB activation, and other aspects of innate immunity. During the next year we plan to continue studies of pyrin biochemistry and gene knockdown, continue investigations of pyrin null mice, continue gene expression studies and immunologic studies of FMF patients both on and off colchicine, and begin to characterize pyrin knockin mice and study a canine model of FMF.